Lyme disease is the most common vector-borne disease in the United States. Understanding the molecular mechanisms by which Borrelia burgdorferi, the etiologic agent, survives in nature will help drive development of innovative strategies for prevention. We have demonstrated that the function(s) of Borrelia iron- and copper-binding protein A (BicA), encoded by bb0690, is critically important for B. burgdorferi survival in an infectious life cycle through the tick and the mammal. We have discovered that iron and copper are among major transition metals in B. burgdorferi and the homeostasis of these metals is altered in a mutant deficient in BicA and in a mutant deficient in a known metal transporter. These data challenge the current dogma in the field - lack of a role for iron in the Lyme disease pathogen. To date, all evidence either for or against the presence of iron in B. burgdorferi is based on in vitro experiments where the spirochetes were grown in artificial media. Therefore, a critical question to be addressed is whether iron and copper play any role in the natural life cycle of this tick-borne pathogen. This is the question we are seeking to address with the experiments outlined in this proposal. Iron and copper are important metal cofactors in many biological reactions, but free iron and copper are toxic to cells due to their abilities to catalyze the Fenton reaction. Hence, organisms utilizing iron or copper have evolved many strategies to control intracellular levels of free iron and copper. Metal-binding proteins such as ferritins and metallothioneins represent one such strategy. Our preliminary data show that (i) BicA is a novel fusion between an iron-binding ferritin-like molecule and a copper-binding metallothionein; (ii) B. burgdorferi lacking BicA has reduced levels of iron and copper, and exhibits a growth defect in media containing iron or copper; and (iii) B. burgdorferi requires BicA for persistence in the tick and subsequent transmission to a new host. Thus, we hypothesize that iron and copper detoxification by BicA is critical to B. burgdorferi survival in the tick and he mammal. To test this hypothesis, we propose to demonstrate that (i) iron and copper are the metals bound by native BicA in B. burgdorferi; (ii) iron- and copper-binding by BicA protects B. burgdorferi against iron and copper detoxification, respectively; (iii) the iron- and copper-bindin activities of BicA are important for B. burgdorferi survival during its infectious life cycle throuh the tick and the mammal. Successful execution of these lines of investigation will not only elucidate the molecular mechanisms underlying B. burgdorferi persistence in nature but also bring about a paradigm change in our understanding of the Lyme disease spirochete.